ABSTRACT
The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.
Subject(s)
Cyclic GMP-Dependent Protein Kinase Type I/chemistry , Animals , Cyclic GMP/chemistry , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinase Type I/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Humans , Immunoblotting/methods , Immunoprecipitation/methods , Protein Binding , Substrate SpecificityABSTRACT
Platelet activation is strongly affected by nitric oxide/cyclic GMP (NO/cGMP) signaling involving cGMP-dependent protein kinase I (cGKI). Previously it was shown that interaction of the cGKI substrate IRAG with InsP(3)RI is essential for NO/cguanosine monophosphate (GMP)-dependent inhibition of platelet aggregation in vitro and in vivo. However, the role of Inositol-trisphosphate receptor associated cGMP kinase substrate (IRAG) for platelet adhesion or granule secretion was unknown. Here, we analysed the functional role of IRAG for platelet activation. Murine IRAG-deficient platelets displayed enhanced aggregability towards several agonists (collagen, thrombin and TxA2). NO- or cGMP-dependent inhibition of agonist induced ATP- or 5-HT secretion from dense granules, and P-selectin secretion from alpha granules was severely affected in IRAG-deficient platelets. Concomitantly, the effect of NO/cGMP on platelet aggregation was strongly reduced in IRAG-deficient platelets. Furthermore, GPIIb/IIIa-mediated adhesion of platelets to fibrinogen could only weakly be inhibited in IRAG-deficient mice contrary to wild-type (WT) mice. Our results suggest that signaling via IRAG is essential for NO/cGMP-dependent inhibition of platelet activation regarding granule secretion, aggregation and adhesion. This platelet disorder might cause that the bleeding time of IRAG-deficient mice was reduced.
Subject(s)
Blood Platelets/metabolism , Cyclic GMP/blood , Membrane Proteins/blood , Nitric Oxide/blood , Phosphoproteins/blood , Platelet Activation/physiology , Animals , Blood Platelets/enzymology , Cyclic GMP-Dependent Protein Kinases/blood , Enzyme Activators/pharmacology , Humans , Mice , Mice, Knockout , Phosphoproteins/deficiency , Phosphoproteins/genetics , Signal TransductionABSTRACT
AIMS: Nitric oxide (NO) and atrial natriuretic peptide (ANP) signalling via cGMP controls smooth muscle tone. One important signalling pathway of cGMP-dependent protein kinase type I (cGKI) is mediated by IRAG (IP(3) receptor associated cGKI substrate) which is highly expressed in smooth muscle tissues. To elucidate the role of IRAG for NO- and ANP-mediated smooth muscle tone regulation, cGKI localization, and for its possible function in blood pressure adjustment, we generated IRAG-knockout mice by targeted deletion of exon 3. METHODS AND RESULTS: IRAG deletion prevented stable interaction of IP(3) receptor type I (IP(3)RI) with cGKIbeta determined by cGMP affinity chromatography. Confocal microscopy in vascular smooth muscle cells (VSMCs) showed that localization of cGKIbeta and cGKIalpha did not change in absence of IRAG. NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic vessels and colon was significantly affected in IRAG-knockout mice. The suppression of cGMP-induced relaxation was not rescued by selective expression of cGKIbeta in smooth muscle from cGKIbeta-transgenic mice. NO-, ANP-, and cGMP-mediated inhibition of the hormone-induced increase in intracellular calcium concentration measured by Fura2 was suppressed in IRAG-deficient VSMC. Telemetric measurements revealed that IRAG-deficient animals exhibited normal basal tone, but were resistant to blood pressure reduction induced by lipopolysaccharide-treatment. CONCLUSION: These findings indicate that signalling of cGKIbeta via IRAG is an essential functional part for regulation of smooth muscle tone and of intracellular calcium by NO (exogenously applicated or endogenously synthesized) and by ANP. IRAG signalling does not modulate basal tone but might be important for blood pressure regulation under pathophysiological conditions.
